Mobile phase dilution scheme for enhanced chromatography

ABSTRACT

A method and device for increasing the loading capacity of a chromatography column through dilution of a mobile phase at the head of the column. A strong mobile phase is provided for dissolving a sample. A sample is injected into the strong mobile phase, and subsequently diluted with a weak mobile phase. The resulting sample-containing weak mobile phase is passed through a chromatography column for retention and separation of the components of the sample.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/414,455, filed Apr. 14, 2003, issued as U.S. Pat. No. 6,790,361,which is a continuation of U.S. application Ser. No. 09/990,636, filedNov. 21, 2001, abandoned, which claims priority to U.S. ProvisionalPatent Application No. 60/252,436, filed Nov. 21, 2000, the contents ofwhich are hereby incorporated herein by this reference.

BACKGROUND

In many fields of science, purified compounds are required for testingand analysis protocols. Purification of a compound involves separatingout a desired component or components from a mixture that containsadditional components or impurities. Chromatography is a method offractionating a mixture to separate components of the mixture. In liquidchromatography, a sample containing a number of components to beseparated is injected into a fluid stream, and directed through achromatographic column. The column is so designed that it separates themixture, by differential retention on the column, into its componentspecies. The different species then emerge from the column as distinctbands, separated in time.

A typical high performance liquid chromatography system (HPLC system) iscomprised of a pump for delivering fluids (the “mobile phase”) at acontrolled flow rate and composition, an injector to introduce a samplesolution into the flowing mobile phase, a tubular column encasementcontaining a packing material or sorbent (the “stationary phase”), and adetector to register the presence and amount of the sample components inthe mobile phase. When the mobile phase is passed through the stationaryphase, each component of the sample will emerge from the column at adifferent time because different components in the sample will havedifferent affinities for the packing material. The presence of aparticular component in the mobile phase exiting the column can bedetected by measuring changes in physical or chemical properties of theeluent. By plotting the detector's signal over time, response “peaks”corresponding to the presence of each of the components of the samplecan be observed and recorded.

Preparative HPLC is a convenient and easy way of isolating and purifyinga quantity of a compound for further studies. Preparative HPLC is notlimited by scale. Depending on the specific application, Preparativeseparations can involve very large columns and sample sizes, resultingin multigram yields, or may be performed using very small columns,resulting in microgram yields. Thus, a common distinction betweenPreparative and Analytical HPLC is that in Preparative HPLC, the sampleis collected after purification, whereas in Analytical HPLC, the samplecomponents are simply detected and quantified.

Typical target requirements for a Preparative separation include highrecovery of the sample compound at a purity exceeding 90%, and a rapid,efficient routine. A single instrument is required to isolate and purifybetween fifty and one hundred samples per day. Therefore, it is highlydesirable to purify and separate the largest possible quantity of samplewith each run, thereby reducing labor, space, operating expense, runtime and associated instrumentation costs.

The combination of Preparative HPLC with a mass spectrometer permits alarge number of samples to be processed automatically. An automatedseparation scheme is guided by mass spectrometer detection. The massspectrometer is set up to detect the expected molecular weight of thetarget sample and direct the collection of the purified component thatcontains this molecular weight.

Other approaches to reduce run time in a Preparative HPLC system includeproviding rapid gradients during gradient elution, and employing ashorter column with smaller particles in the stationary phase. However,these techniques often compromise the results of the analysis, leadingto loss of resolution and a deterioration of peak shape, indicatingdecreased purity of the separated sample.

In Preparative chromatography, it is also desirable to maximize thequantity of sample to be separated per volume of packing material in thecolumn. Smaller volume columns contain less packing material, whichoften will have a significant impact on the cost of the column. However,the resolution between response peaks in a chromatographic analysis or“run” depends, in part, on the loading capacity of the column.Chromatography results are limited by the loading capacity of thecolumn, defined as a threshold for the maximum volume and/or mass ofsample that may be loaded onto the column without compromising results.

The loading capacity for a column can be exceeded in two ways: volumeoverload; and mass overload. Volume overload can be defined as thevolume of injected sample solution where loss of resolution occurs. Massoverload can be defined as the mass of solute in the sample solutionabove which loss of resolution occurs.

Loading capacity of a column can be measured by injecting aprogressively larger amount of sample. Often times, compounds will elutewith multiple peaks having different retention times. A load thatexceeds the column capacity is characterized by a deterioration of peakshape and loss of resolution in the resulting separation.

The column can be any chromatographic column, either of conventional orcartridge design. The column can also be composed of two or more columnsthat are interconnected in some way. An example of such an arrangementwould be the use of an easily replaceable guard column connected inseries upstream from another column, thereby protecting the main columnfrom premature failure due to fouling.

Neue et al. (Advances in Chromatography, 41: 93-136 (2001), thedisclosure of which is hereby incorporated herein by reference) describetechniques for optimizing a reversed-phase gradient separation byvarying the column length, particle sizes and running conditions for theseparation. The article further describes optimized sample loadingschemes for providing simplified and automatable preparative gradientelution.

It is desirable to enhance the loading capacity of a column, therebyallowing for the purification and isolation of a larger quantity of apurified sample per chromatographic run. An increased loading capacityfor a chromatography column also implies less run time required, andlower cost associated with the isolation of a fixed quantity of sample.

SUMMARY OF THE INVENTION

The present invention relates to liquid chromatography instrumentationand solvent delivery systems, and more particularly to a method andapparatus for increasing the loading capacity of a chromatography columnthrough dilution of a mobile phase at the head of the column.

According to one aspect, the present invention provides an enhancedmethod of separating a sample in a chromatography system, comprisinginjecting a sample solution into a strong mobile phase to form asample-containing strong mobile phase, diluting the sample-containingstrong mobile phase with a weak mobile phase upstream from thechromatography column to form a sample-containing weak mobile phase, andpassing the sample-containing weak mobile phase through a chromatographycolumn

According to another aspect, a chromatography system is provided. Thechromatography system comprises a first fluid pump for providing astrong mobile phase at a predetermined flow rate and composition, aninjector for injecting a sample solution into the strong mobile phase toform a sample-containing strong mobile phase, a second fluid pump forproviding a weak mobile phase to the sample-containing strong mobilephase at a predetermined flow rate and composition to form asample-containing weak mobile phase, a chromatographic column containinga chromatographic sorbent for separating the sample and a column fittingdevice for combining the sample-containing strong mobile phase with theweak mobile phase to form the sample-containing weak mobile phase priorto the chromatographic sorbent.

According to another aspect, a column fitting device for achromatographic column is provided. The column fitting device comprisesa first conduit configured to connect with a weak mobile phase flowpath, a second conduit configured to connect with a sample-containingstrong mobile phase flow path, and a third conduit configured to connectwith a chromatographic column. The column fitting device mixes asample-containing strong mobile phase, comprising a sample and a strongmobile phase, with a weak mobile phase to form a sample-containing weakmobile phase, and passes the sample-containing weak mobile phase to thecolumn.

According to another aspect, a chromatographic column is provided. Thecolumn comprises a chromatographic sorbent bed, a common inlet fluidpath upstream from the sorbent bed, a first inlet fluid connection portconfigured to connect with the common inlet fluid path, and a secondinlet fluid connection port configured to connect with the common inletfluid path.

In another aspect, a solvent delivery subsystem for a chromatographysystem is provided. The subsystem comprises a first pump for providing astrong mobile phase, an injector for injecting a sample into the strongmobile phase to form a sample-containing strong mobile phase, a secondpump for providing a weak mobile phase and a column fitting device formixing the weak mobile phase with the sample-containing strong mobilephase to form a sample-containing weak mobile phase

According to another aspect, a packaged column fitting device isprovided. The packaged column fitting devices comprises a fluid conduithaving a first conduit configured to attach to a weak mobile phase flowpath, a second conduit configured to connect with a sample-containingstrong mobile phase flow path and a third conduit configured to connectwith a chromatographic column. The column fitting device is packagedwith instructions for use with an enhanced method of separating a samplein a chromatography system.

According to another aspect, a packaged pump for a chromatography systemis provided. The packaged pump is configured to provide a weak mobilephase to a sample-containing strong mobile phase at a predetermined flowrate to dilute the sample-containing strong mobile phase. The pump ispackaged with instructions for use with an enhanced method of separatinga sample in a chromatography system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a traditional instrument configuration fora solvent delivery system in a Preparative chromatography system.

FIG. 2 is an illustration of an instrument configuration for a solventdelivery system in a Preparative chromatography system according to theteachings of the present invention, utilizing a single solvent loadingpump together with a multisolvent gradient delivery pump.

FIG. 3 is an illustration of a column fitting used in accordance withthe teachings of the present invention to increase the loading capacityof a chromatographic column.

FIG. 4 is a detailed drawing of a chromatographic column with multipleinlet ports for providing at column dilution of a mobile phase accordingto the teachings of the invention.

FIG. 5 is an illustration of an alternate instrument configuration for asolvent delivery system in a Preparative chromatography system accordingto the teachings of the present invention, where the chromatographiccolumn is comprised of a guard column with multiple inlet portsconnected in series with another chromatographic column.

FIG. 6 a is a chromatogram illustrating a separation of a 500-μL-samplesolution for a standard chromatography system.

FIG. 6 b is a chromatogram illustrating a separation of a 2000 μL samplesolution for a standard chromatography system.

FIG. 7 a is a chromatograph illustrating a separation of a 500 μL samplesolution using the chromatography system of FIG. 2.

FIG. 7 b is a chromatogram illustrating a separation of a 1000 μL samplesolution using the chromatography system of FIG. 2.

FIG. 7 c is a chromatogram illustrating a separation of a 2000 μL samplesolution using the chromatography system of FIG. 2.

FIG. 7 d is a chromatogram illustrating a separation of a 4000 μL samplesolution using the chromatography system of FIG. 2.

FIG. 8 a is a chromatogram illustrating a separation using the columnfitting device and column of FIG. 3.

FIG. 8 b is a chromatogram illustrating a separation using a columnhaving multiple inlet ports as illustrated in FIG. 5.

DETAILED DESCRIPTION OF THE INVENTION

An illustrative embodiment of the present invention provides an enhancedchromatographic system and method for separating and purifying a sample.The illustrative embodiment will be described below relative to animplementation in a Preparative HPLC system. Those skilled in the artwill appreciate that the present invention may also be implemented onother types of chromatography systems. The illustrative embodimentenhances the loading capacity of a chromatography column through adilution scheme.

The present invention provides an enhanced chromatography methoddesigned to increase the loading capacity of a chromatography column andenhance the separation of a sample in a Preparative HPLC system. In anillustrative embodiment of the present invention, an at-column dilutionscheme is implemented into a chromatography system to dilute a strongmobile phase containing the sample prior to introduction of the sampleonto the chromatographic column. This dilution has the effect ofincreasing the loading capacity of a column, thereby allowing a greaterquantity of sample to be separated during a chromatographic run.Alternatively, a shorter column may be used to separated a fixed amountof sample, thereby reducing the run time required to separate the fixedamount of sample.

According to the illustrated embodiment, a sample solution is injectedinto a strong mobile phase. The sample solution may be comprised of amixture of compounds dissolved in a relatively small volume of solvent,having good solubility for the sample components. One of ordinary skillin the art will recognize that any suitable solvent may be used,depending on the sample, the solubility of the sample, and other relatedparameters. A common example of a sample solution would be a mixture ofdrug compounds dissolved in dimethylsulfoxide (DMSO) or other suitablesolvent, such as dimethylformamide (DMF), tetrahydrofuran (THF) andmixtures thereof.

The injection of the sample forms a sample-containing, strong mobilephase that is subsequently diluted at the head of a chromatographycolumn by the addition of a weak mobile phase. The resulting solution, asample-containing weak mobile phase, is introduced into a chromatographycolumn. The dilution of the sample-containing strong mobile phase at thehead of a column has the effect of increasing the loading capacity ofthe column, thereby allowing a greater quantity of sample to beseparated during a chromatographic run.

In chromatography, the mobile phase is a fluid chosen to dissolve thesample solution and carry the sample through the stationary phase of thechromatography system. As used herein, mobile phases are termed “strong”or “weak” in relation to each other. The “strength” (e.g., strong orweak) of a mobile phase refers to the elution strength of the mobilephase and is used to describe the affinity that a sample component willhave for either the mobile phase or stationary phase.

The terms “strong mobile phase” and “weak mobile phase” are known in theart. As used herein, a “strong mobile phase” refers to a mobile phasethat has a high elution strength and results in little or no retentionof the sample on the chromatographic sorbent. A sample dissolved in a“strong” mobile phase will have a greater affinity for the mobile phasethan the stationary phase, resulting in little or no retention of thesample on the stationary phase and a shorter elution time.

As used herein, a “weak mobile phase” refers to a mobile phase that hasa low elution strength and results in higher amount of retention of thesample on the chromatographic sorbent relative to a strong mobile phase.Contrary to a sample dissolved in a strong mobile phase, a sampledissolved in a “weak” mobile phase will have less affinity for themobile phase than the stationary phase, resulting in sample componentsbeing strongly retained on the stationary phase and a longer elutiontime.

In order for the sorbent bed to preferentially retain sample components,the mobile phase is, at least initially, advantageously of low tointermediate strength. Otherwise the sample components will simply passthrough the column with little or no retention or separation. InGradient Preparative HPLC, the initial mobile phase strength is keptquite low, so that sample components are highly retained on the sorbentbed. The mobile phase strength is then systematically increased overtime to elute in turn each sample component. This approach places asolubility limit on the volume and/or mass of sample solution which canbe injected into the HPLC.

Depending on the retention mechanism being used, many different mobilephase properties can be used to adjust mobile phase strength. Inparticular, the solvents are selected so as to adjust the strength ofthe mobile phase. The types of solvents used are well known to thoseskilled in the art. For example, in both “Reversed Phase” and “NormalPhase” chromatography, the ratio of organic solvent to water in themobile phase is typically modified to adjust the strength of the mobilephase. In the case of an ion-exchange-based separation, mobile phase pHand/or ionic strength is commonly manipulated to adjust the strength ofthe mobile phase. One skilled in the art will recognize that althoughthe examples provided in the present invention focus on “Reversed Phase”separations, the enhanced technique of the present invention can beadapted to other separation modes as well.

In Reversed Phase Chromatography, a non-polar stationary phase is usedin conjunction with more polar, largely aqueous mobile phases. Becausesample retention in this case is driven by hydrophobic interaction, astrong mobile phase, i.e., one which can easily elute the sample fromthe stationary phase, will be one having a high percentage of organicsolvent. Conversely, a weak mobile phase will have a lower percentage oforganic solvent in Reversed Phase Chromatography.

In Normal-Phase Chromatography, the stationary phase is more polar thanthe mobile phase. A common application of Normal-Phase Chromatography isseen in the use of a polar stationary phase, such as silica or alumina,with a mobile phase having a high percentage of organic solvent. InNormal-Phase, a weak mobile phase would have a high percentage oforganic solvent, while a strong mobile phase would have a lowerpercentage of organic solvent.

In Ion-Exchange Chromatography, retention of the sample on thestationary phase is controlled through the interaction of chargedanalytes with oppositely charged functional groups on the stationaryphase surface. Because both the sample components and the stationaryphase could contain either cation or anion exchange groups (and possiblyboth) these separations are strongly influenced by changes in mobilephase pH and/or ionic strength. In the case of ion-exchange separations,raising or lowering the pH and/or ionic strength of the mobile phaseresults in either an increase or a decrease in the elution strength ofthe mobile phase, depending on the pKa of the sample and whether thestationary phase is a cation or anion exchanger. The pH and/or the ionicstrength may be raised or lowered as the separation requires, therebyadjusting the elution strength of the mobile phase. A large applicationarea is the separation of biopolymers, specifically proteins andpeptides.

Changes in the temperature of the mobile phase can also influence sampleretention in many modes of chromatography. Generally, as the temperatureof a mobile phase is increased, the mobile phase strength is alsoincreased, resulting in shorter retention time on the stationary phase.Although the effect is not often as dramatic as seen with changes inpercentage of organic solvent or pH, it can generally be observed acrossall modes of chromatography. This effect can be a result of simplyincreasing the solvating power of a mobile phase at elevatedtemperature, or of temperature-induced changes to the molecularstructure of the sample molecules in solution. A common application areais the separation of samples having complex structure, such as proteinsand related biopolymers.

In accordance with the present invention, a strong mobile phasedissolves the sample. The strong mobile phase comprises a strong organicsolvent, such as, e.g., 100% acetonitrile, in which the sample has goodsolubility. A strong mobile is capable of dissolving a larger quantityof a sample than a weak mobile phase. As used herein, the term“sample-containing strong mobile phase” consists of the strong mobilephase and the dissolved sample carried in the strong mobile phase.

Although a strong mobile phase is suitable for dissolving a largequantity of a sample, the strength of this mobile phase causes thesample components to be poorly retained by the sorbent bed, leading topeak distortion and degraded chromatographic separation. To regulate thestrength of the solvent prior to passing the sample-containing strongmobile phase through the column, the sample-containing strong mobilephase is diluted by adding a weak mobile phase to the sample-containingstrong mobile phase to form a sample-containing weak mobile phase. The“sample-containing weak mobile phase” includes the strong mobile phase,the sample and the weak mobile phase (i.e., the diluent). Thesample-containing weak mobile phase is weaker in elution strength thanthe strong mobile phase, in that the sample has lower affinity for themobile phase and therefore a higher affinity for the stationary phase.The diluted sample-containing mobile phase is suitable for carrying thesample onto the stationary phase without degrading the chromatographicresults.

The above-described technique is capable of significantly increasing theloading capacity of a column. This technique enhances conditions forboth dissolving a sample and carrying the sample through achromatography column. In this manner, a larger quantity of sample maybe analyzed in a single chromatography run. A chromatography systemdevoid of the described dilution scheme is limited to a smaller amountof sample that may be separated without degrading the end result.Application of the enhanced method of separating a sample using theabove-described dilution scheme can increase the loading capacity of acolumn. Preferably, the loading capacity of the column is increased byat least about two-fold, and more preferably between about two-fold andabout 80-fold, and still more preferably by at least five-fold;ten-fold; 20-fold; 30-fold; 40-fold; 50-fold; 60-fold; 70-fold; and80-fold.

In addition to increasing the loading capacity of a column, the enhancedchromatography method results in improvement in peak shape andresolution for a fixed quantity of sample. When a sample is injectedinto a solvent in which the sample has insufficient solubility, sampleprecipitation results. This causes troublesome blocking inside injectionports, valves and interconnecting tubing lines. However, a solvent withhigh sample solubility also leads to poor results, as it tends to carrythe sample too far down the length of the column, resulting in unwantedpeak broadening and poor separation efficiency. The chromatographymethod of the present invention reduces precipitation of the sample ininjection ports, valves, and interconnecting tubing lines, while alsoallowing a strong retention of the sample at the head of thechromatography column to improve separation of the different components.By initially providing a strong mobile phase to fully dissolve thesample, and subsequently diluting the mobile phase before introductiononto the column, separation and purification of a sample are enhanced.

In a traditional Preparative HPLC system, illustrated in FIG. 1, asingle gradient pump 11 delivers a variable strength mobile phase ofcontrolled composition to the system. The sample solution to beseparated is inserted through an injector loop 12 into the stream ofsolvent, and delivered to the chromatography column 13 as a slug ofsample solution sandwiched in the stream of mobile phase. The mobilephase is forced through the column 13 and passed to a detector 14 forevaluation. Individual sample components are retained to varying degreeson the sorbent bed, and elute in turn from the column over the course ofthe chromatographic run.

In a Gradient Preparative HPLC the mobile phase solvent strength isvaried over the course of the run, in order to achieve the desiredseparation. An initial low strength mobile phase flowing through thecolumn causes the sample components to be strongly absorbed on thesorbent within the column. After the sample is loaded on the column 13,a gradient is initiated to increase the strength of the mobile phase.The gradient, effected by the gradient pump 11, causes the individualcomponents of the sample to be sequentially eluted from the column 13.

Other separation conditions may require that the mobile phasecomposition remain fixed over time (this mode being termed “IsocraticPreparative HPLC”). In this case, the mobile phase strength remainsfixed at an intermediate level where all of the sample components arepartially retained by the sorbent.

There are inherent limitations to the traditional Preparative HPLCsystem illustrated in FIG. 1. It is desirable to maximize the amount ofcompound that may be purified and isolated in a chromatography run. Aninitial approach would be to load an increased volume of a samplesolution, comprised of a sample dissolved in a suitable solvent.However, it has been found that a large volume of sample solution, wheninjected into the system, degrades chromatography and creates peakdistortions due to volume overload. Therefore, it would be desirable toload the largest amount of compound in a small volume of solution tolimit the total injection volume. This results in a high sampleconcentration. High sample concentrations require a strong solvent.However, the strong solvent also degrades the separation and createspeak distortions due to mass overload. Therefore, solubility of a samplein the mobile phase of the system illustrated in FIG. 1 is limited.These conflicting principles must be resolved in order to provideoptimal separation of a large amount of compound in a chromatographysystem, without excessive mass or excessive volume overload.

The present invention overcomes these limitations by increasing theapparent loading capacity of a column to maximize the amount of samplethat can be separated per run cycle. Thus, the present invention, asillustrated in FIG. 2, provides a chromatographic column 25 forseparating a sample, a loading pump 22 for providing a strong mobilephase, such as, e.g., acetonitrile, to the system and a separategradient pump 21 for providing a variable strength, weak mobile phaseflow to the system and a fitting 24 in communication with the column 25for combining the outputs of the loading pump 22 and the gradient pump21. The loading pump 22 delivers a stream of a strong mobile phasethrough the injector 23. A sample is first dissolved in a solvent andthen injected through the injector 23 into the strong mobile phasestream to form a sample-containing strong mobile phase. In this case, ahigh concentration of sample in a small volume of solution is permitted.The sample is carried to the fitting 24 as a slug sandwiched in thestrong mobile phase stream. At the fitting 24, the sample-containingstrong mobile phase, comprised of the sample solution and high strengthmobile phase, is diluted with a weak mobile phase flow from the gradientpump 21 to form a sample-containing weak mobile phase. Thesample-containing weak mobile phase is then passed through the fitting24 to the column 25. The diluted, sample-containing weak mobile phase,comprised of the sample solution and the combination of the mobilephases from the gradient pump and the loading pump, is passed throughthe column 25 and subsequently conveyed to detectors 26 for evaluationand collection of the purified compound.

In a preferred embodiment, the initial flow rate of the weak mobilephase from the gradient pump 21 is greater than the initial flow rate ofthe strong mobile phase; advantageously, the initial flow rate of theweak mobile phase is at least about twenty times the initial flow rateof the strong mobile phase from the loading pump 22. In this manner, thediluted mobile phase at the head of the column contains 5% of the strongmobile phase and 95% of the weak mobile phase, which is similar to themobile phase at the head of the column in the system of FIG. 1 at thestart of a gradient separation.

In an alternate embodiment of the present invention, two single solvent,or non-gradient pumps can be used, where one pump delivers only thestrong mobile phase flow, and a second pump delivers only the weakmobile phase flow. The system is identical to the system shown in FIG.2, with the exception that the gradient pump 21 is replaced with asingle solvent isocratic pump that delivers a weak mobile phase of fixedcomposition. In this embodiment, the mobile phase composition enteringthe column is controlled by flow programming of the two single solventpumps. A potential advantage of this embodiment may be in lowerinstrumentation cost associated with the gradient pump 21.

A suitable fitting 24 used for the above-described mobile phase dilutionscheme is illustrated in FIG. 3. The fitting 24 is comprised of threefluid conduits having a common flow path. A flow path 31 delivers thesample-containing strong mobile phase to a junction point 32. A dilutionflow path 33 delivers the weak mobile phase to the junction point 32. Atthe junction point 32, the sample-containing strong mobile phase iscombined and diluted with the weak mobile phase to form asample-containing weak mobile phase. This sample-containing weak mobilephase passes through the third conduit 34 and onto the head of thecolumn (25 in FIG. 2).

The junction point 32 may comprise a cavity having any suitable size andshape for mixing the two flow paths. The junction point 32 may furtherinclude a mixing element to facilitate dilution and mixing of asample-containing strong mobile phase with a weak mobile phase.

According to the embodiment shown in FIG. 3, the fitting 24 isinterposed between a chromatography column, a gradient pump and aloading pump. The fitting may be connected to the inlet of thechromatographic column and the outlets of the gradient pump and theloading pump through any suitable means known in the art. For example,one or more of the ends of the conduits 31, 33 or 34 may be threaded tofacilitate connection with the loading pump, the chromatographic columnand/or the gradient pump, respectively. Alternatively, the conduits maybe connected to the inlet of the chromatography column and/or theoutlets of the gradient pump and the loading pump by friction-fit. Theconduits 31, 33, 34 forming the fitting 24 may have any suitable size,and shape, depending on the particular application.

An alternate design for the column fitting is illustrated in FIG. 4. InFIG. 4, the column fitting for combining two flow paths and deliveringthe combined flow path to a column is integrally formed on achromatographic column 40. The column body 41 is a tube of variouslength, diameter, and material chosen for the intended use and is packedwith a chromatographic sorbent 42 which may be of various particle size,shape, or chemical composition. The bed is contained at both inlet andoutlet ends by porous filter elements 43, 44, that have been chosen toretain the stationary phase. The filter elements may be sealed in placeusing a number of techniques, including press fitting into either thecolumn end fitting or column body. A column outlet end fitting 45attaches the outlet filter 44 to the column body 41, and includes afluid connection port 46 for the fluid exiting the column. A columninlet end fitting 47 attaches the inlet filter 43 to the column body 41,and provides multiple connection ports 48, 49 for fluid streams enteringthe column. In a preferred embodiment, the first connection port 48delivers a sample-containing strong mobile phase to the column and thesecond connection port 49 delivers a weak mobile phase to the column.The fluid streams are then mixed in close proximity to thechromatographic bed 42 to dilute the sample-containing strong mobilephase.

In an alternate design, the fluid streams entering the column arecombined in a recessed cavity 50 located within the inlet end fitting,where they are acted on by a mixing element 51, which is inserted intothe cavity 50. The mixing element may be of any conventional design.Once mixed, the diluted sample-containing mobile phase passes throughthe inlet filter and the chromatographic bed, and then eventually exitsthe column. In a preferred embodiment, the column can be of a cartridgesystem design, consisting of a cartridge holder, which includes thepreviously described fluid connection ports, and mixing elements, and aremovable cartridge type column, which contains the packedchromatographic sorbent bed and retaining porous filter elements.

An embodiment of the complete Preparative system of an illustrativeembodiment of the present invention is illustrated in FIG. 5. A loadingpump 53 provides a controlled flow of strong mobile phase through aninjector 54 and into the head of a multiple inlet guard column 55through a first inlet. A gradient pump 52 provides a controlled flow ofvariable strength, weak mobile phase to a second inlet of the guardcolumn 55. The fluids entering the column 55 are mixed in closeproximity to the sorbent bed and are passed onto the chromatographicsorbent contained within columns 55 and 56 for separation and eventualdetection by a detector 57.

The dilution scheme of the present invention enhances chromatographicresults significantly. A strong initial mobile phase for dissolving asample permits loading of a large amount of a sample in a small volume.This maximizes dissolution of the sample in the mobile phase. Bysubsequently diluting the sample-containing strong mobile phase prior tointroduction of the sample onto the column, optimum separationconditions are achieved. The illustrative dilution scheme resolves theconflicting principles of both a large injection volume and a strongsample solvent degrading chromatography.

An enhanced loading capacity provides significant advantages forreducing the time required to purify and isolate a quantity of compound,and therefore the operating costs, of a separation. For a given samplesize, a shorter column may be used, leading to a reduced run time, lowersolvent consumption, and reduced column expense while producinghigh-quality results. Therefore, an increased number of samples, may bepurified on a single chromatography system per day. For a given columnsize, a larger load of sample can be purified during each run, therebydecreasing the number of runs required to purify a specific quantity ofsample.

In addition, the implementation of the dilution scheme of the presentinvention provides increased resolution, and improved peak shape for agiven mass of sample, thereby enhancing the purity of collected peaksThese effects are illustrated in the non-limiting examples below.

EXEMPLIFICATION OF THE INVENTION Example 1 The Change in SeparationQuality with Increasing Injection Load Using a Standard ChromatographicMethod

A sample mixture is prepared by combining Diphenhydramine, Oxybutynin,and Terfenadine, at a concentration of 20 milligrams per milliliter foreach component, using dimethylsulfoxide (DMSO) as the sample solvent. Astandard chromatographic system as described in FIG. 1 is used toseparate the sample, where the column 13 is comprised of a 19 mminternal diameter×10 mm long guard column of conventional designconnected in series with a 19 mm internal diameter×30 mm long column ofconventional design. The chromatographic columns are packed withstandard reversed phase C18 bonded sorbent (Waters' XTerra MS-C18, 5micron particles). A linear gradient elution profile is used, with astarting mobile phase composition of 5% acetonitrile/95% watercontaining ammonium bicarbonate pH 10 buffer, and a final mobile phasecomposition of 90% acetonitrile/10% water containing ammoniumbicarbonate pH 10 buffer. The mobile phase composition profile versustime is set forth in Table 1 below. Flow rate is set to 30 millilitersper minute, and a UV detector of conventional design is used to monitorthe column effluent.

FIGS. 6 a and 6 b show separation results for 500 μL and 2000 μL sampleinjection volumes, respectively, using the standard chromatographysystem of FIG. 1. As the injection load increases from 500 μL to 2000μL, the baseline resolution between peaks is no longer achieved, andpeaks 1 and 3 split to form multiple peaks. The loss of resolution andthe appearance of split peaks in FIG. 6 b result from exceeding theloading capacity of the chromatographic column.

TABLE 1 Time Water Acetonitrile 100 mM NH₄HCO₃, pH 10 Standard System AsUsed In Example 1 0.00 90 5 5 0.50 90 5 5 0.60 55 40 5 4.00 5 90 5 4.305 90 5 4.40 90 5 5 6.00 90 5 5 At-Column Dilution System As Used InExample 2 and 3 0.00 90 5 5 2.50 90 5 5 2.60 55 40 5 6.00 5 90 5 6.30 590 5 6.40 90 5 5 8.00 90 5 5

Example 2 The Change in Separation Quality with Increasing InjectionLoad Using the Enhanced Chromatographic Method of the IllustrativeEmbodiment of the Present Invention

A chromatographic system is used as shown in FIG. 2, where the column25, and detector 26 are identical to those described in Example 1. Afitting device 24 (as shown in FIG. 3) is placed in front of column 25.A linear gradient elution profile is delivered from gradient pump 21 ata flow rate of 28.5 milliliters per minute. The loading pump 22 is setto a flow rate of 1.5 milliliters per minute, resulting in a totalcombined flow rate of 30 milliliters per minute, similar to the firstExample. The gradient composition over time is set forth above inTable 1. The same gradient composition is used as in Example 1, however,an initial hold is added to the gradient to allow pumping of the loadingsolvent from the loading pump 22 through the injector 23, therebyensuring that the sample is completely transferred to the column beforethe gradient starts. The same sample mixture is injected onto the systemas used in Example 1.

FIGS. 7 a, b, c, & d show separation results for 500, 1000, 2000, and4000 μL sample injection volumes, respectively. As the injection loadincreases, it can be seen that the baseline resolution between peaks ismaintained, with excellent baseline resolution and no occurrence ofsplit peaks. As shown, the use of the column fitting device to dilutethe sample-containing strong mobile phase prior to introduction to thecolumn significantly increases the loading capacity of the volume, whencompared with a standard chromatography system that does not use thedilution scheme, as described in Example 1.

Example 3 A Comparison Between the Use of the Embodiment of FIG. 3 andthe Embodiment of FIG. 4

A chromatographic system is arranged as shown in FIG. 5. Theconfiguration of FIG. 5 is identical to that used in Example 2, with theexception that the guard column 55 contains an integral dual inletdesign (as shown in FIG. 4) to provide the mobile phase dilution schemeof the illustrative embodiment. The integral dual inlet design is usedin place of the fitting device 24 in Example 2. The sample composition,gradient elution profile, and all mobile phase flow rates are identicalto those used in Example 2.

FIGS. 8 a and b compares separation results for a 4000 μL injection ofsample solution using the chromatographic methods and systems describedin Examples 2 and 3, respectively. The results show that the columnhaving an integral multiple inlet design, as set forth in Example 3,performs in a substantially similar manner to the chromatographicfitting device of Example 2.

Although the invention has been described in detail with reference to anillustrative embodiment and application, those skilled in the art willappreciate that various modifications and variations may be made withoutdeparting from the intended scope of the present invention as defined inthe appended claims.

1. A chromatography system comprising: a first fluid pump for providinga strong mobile phase at a predetermined flow rate and composition, aninjector for injecting a sample solution into the strong mobile phase toform a sample-containing strong mobile phase, a second fluid pump forproviding a weak mobile phase to the sample-containing strong mobilephase at a predetermined flow rate and composition to form asample-containing weak mobile phase, a chromatographic column containinga chromatographic sorbent for separating the sample, and a columnfitting device for combining the sample-containing strong mobile phasewith the weak mobile phase to form the sample-containing weak mobilephase prior to the chromatographic sorbent; wherein the column fittingdevice is integral with an inlet end fitting of a chromatographiccolumn, said column fitting device having a recessed cavity locatedwithin the inlet end fitting such that the fluid streams entering thecolumn fitting device are combined in the recessed cavity.
 2. The systemof claim 1, wherein the column fitting device comprises a first conduitconnected to the first fluid pump, a second conduit connected to thesecond fluid pump and a third conduit connected to an inlet end of thechromatographic column.
 3. The system of claim 2, wherein the firstconduit includes a connecting mechanism to connect the fluid fittingdevice to the first fluid pump.
 4. The system of claim 2, wherein thesecond conduit includes a connecting mechanism to connect the fluidfitting device to the second fluid pump.
 5. The system of claim 1,wherein the weak mobile phase has a first flow rate and the strongmobile phase has a second flow rate, and the first and second flow ratesvary after the sample reaches the column.
 6. In a chromatography system,a solvent delivery subsystem comprising a first pump for providing astrong mobile phase; an injector for injecting a sample into the strongmobile phase to form a sample-containing strong mobile phase; a secondpump for providing a weak mobile phase; a column fitting device formixing the weak mobile phase with the sample-containing strong mobilephase to form a sample-containing weak mobile phase; and achromatographic column containing a chromatographic sorbent forseparating the sample, wherein the column fitting device is integralwith an inlet end fitting of a chromatographic column, said columnfitting device having a recessed cavity located within the inlet endfitting such that the fluid streams entering the column fitting deviceare combined in the recessed cavity.